ANNALS OF HEPATOLOGY
Volume
3
Number
4
October-December
2004
Artículo:
Liver aquaporins: Significance in
canalicular and ductal bile formation
Copyright © 2004:
Mexican Association of Hepatology
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130
Annals of Hepatology
2004; 3(4):3(4)
October-December:
130-136 MG
Annals of Hepatology
2004: 130-136 MG
Concise Review
Annals
of
Hepatology
Liver aquaporins: Significance in canalicular
and ductal bile formation
Raúl A. Marinelli;1 Sergio A. Gradilone;1 Flavia I. Carreras;1 Giuseppe Calamita;2
Guillermo L. Lehmann1
Abstract
Bile is primarily secreted in hepatocytes (i.e. the canalicular bile) and subsequently delivered to the intrahepatic bile ducts, where is modified by cholangiocytes
(i.e. the ductal bile). Bile formation is the result of the
coordinated interactions of membrane-transport systems that generate the vectorial movement of solutes
and osmotically driven water molecules. Hepatocytes
and cholangiocytes express aquaporins, specialized
membrane channel proteins that facilitate the osmotic
transport of water. In this review, we provide a summary of what is known on liver AQPs and their significance in canalicular and ductal bile formation under
normal and pathological conditions.
Key words: Water channels, hepatocytes, cholangiocytes.
Primary bile is secreted by hepatocytes at the bile
canaliculus and is subsequently modified in composition
and volume by cholangiocytes, the cells that line the intrahepatic bile ducts.1 Bile consists of about 95% water
and water transport by liver cells is thought to occur passively in response to local, transient, osmotic gradients
generated by the active transport of certain solutes. Both
1
Instituto de Fisiología Experimental, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, 2000 Rosario, Santa Fe, Argentina.
2
Dipartimento di Fisiologia Generale ed Ambientale, Università degli
Studi di Bari, 70126 Bari, Italy.
Address for correspondence:
Raúl A. Marinelli, Ph.D.
Instituto de Fisiología Experimental. Facultad de Ciencias Bioquímicas y Farmacéuticas. Universidad Nacional de Rosario. Suipacha 570,
2000 Rosario, Santa Fe, Argentina. Phone: 54-341-4305799. Fax: 54341-4399473.
E-mails: [email protected] / [email protected]
hepatocytes and cholangiocytes express aquaporins
(AQPs),2,3 a family of membrane channel proteins that facilitate the osmotically driven movement of water molecules.4 There is compelling experimental evidence suggesting that AQPs are key players in bile formation.
Aquaporin water channels
The first AQP was identified in erythrocytes by Dr. Peter Agre from Johns Hopkins University, U.S.A.5,6 who
was awarded with the Nobel Prize in Chemistry 2003.
AQPs are a family of integral homotetrameric membrane
proteins widely distributed in mammals, plants, and lower
organisms. Eleven mammalian aquaporin proteins, numbered 0 to 10, have been identified thus far.7 Each aquaporin consists of four independent channels assembled into a
functional unit. Primary sequence of AQPs revealed that
each subunit is composed of approximately 270 amino acids with cytoplasmic carboxy- and amino-terminal ends.
They consist of six bilayer-spanning domains connected by
three extracellular (A, C, and E) and two intracellular loops
(B and D). Two of these loops enclose the conserved motif
Asn-Pro-Ala (NPA) which is part of the aqueous pore4
(Figure 1). Several recent studies have revealed the highresolution structures of the water channel protein family
that explain their selectivity. The three key features for
channel selectivity are:8 (i) size restriction. The pore narrows to a diameter of 2.8 Å (approximately the diameter of
a water molecule); (ii) electrostatic repulsion. The residue
Arg-195 imposes a barrier to cations, including protonated
water (H3O+); (iii) water dipole reorientation. Positively
charged dipoles reorient water molecules and prevent the
formation of a proton conductance.
Aquaporins are characteristically inhibited by mercurials. The inhibitory site corresponds at the Cys-189, proximal to the NPA motif in loop E.9 Certain AQPs (e.g.,
AQP4) lack the cysteine at this site leading to the lack of
inhibition by mercurials.10,11 AQPs work primarily as water-transporting channels, although some of them (e.g.,
AQP9) are also permeable to certain small solutes such as
glycerol and urea.12
AQPs have been shown to be expressed in many epithelial cells; typically, one or more specific AQPs exist in
a particular water-transporting epithelial cell. They are either constitutively expressed or regulated, often by agonist-induced trafficking from an intracellular vesicular
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Abbreviations: AQP, aquaporin; Bsep, bile salt export pump; Mrp2,
multidrug resistance-related protein 2; CFTR, Cystic Fibrosis
Transmembrane Regulator; AE2, Cl-/HC03- anion exchanger 2.
This work was supported by Grant PICT 05-10590 (to R.A. Marinelli)
from Agencia Nacional de Promoción Científica y Tecnológica, and by
Grant PIP 03020 (to R.A. Marinelli) from Consejo Nacional de
Investigaciones Científicas y Técnicas (CONICET).
RA Marinelli et al. Liver aquaporins
A
A
C
N
Extracellular
PA
B
H2O
E
1
H2N
2
N
3
B
P
4
5
6
Lipid bilayer
5
D
A
COOH
6
Intracellular
H2O
Table I. Liver aquaporin proteins.
Name
Transport
function Cell type
Subcellular
localization
vesicles
Regulated
trafficking References
AQP 0
water
hepatocyte
no
[14]
AQP 1
water
AQP 4
AQP 8
water
water
AQP 9
water,
glycerol,
urea, other
solutes
cholangiocyte vesicles, secretin, cAMP [32,33]
APM
peribiliary
PM
no
[36]
endothelium
cholangiocyte BLPM
no
[34]
hepatocyte (*) vesicles,
glucagon,
CPM
cAMP
[15-18]
hepatocyte
BLPM
no
[14,19,20]
CPM, canalicular plasma membranes; APM, apical plasma membranes; BLPM,
basolateral plasma membranes; PM plasma membranes.
(*) it was recently reported the presence of AQP8 mRNA in cholangiocytes (Ref. 35)
compartment to the plasma membrane allowing for rapid
changes in membrane permeability, depending upon
physiological needs. An example of this is the translocation of AQP2 in response to vasopressin in the tubule epithelia of the collecting duct of the kidney.13
Five members of the mammalian aquaporin family
were found to be expressed in liver cells both at mRNA
and protein level (Table I).
Hepatocyte aquaporins
It has been recently shown that rat hepatocytes express
mRNA and protein for AQP0,14 AQP8,15-18 and
AQP9.14,19,20
Aquaporin-8 (AQP8)
131
Figure 1. Membrane topology and
organization of the aquaporin water
channel molecule. A: A single AQP
monomer is composed of six transmembrane domains connected by
loops A to E, with two NPA boxes
and with the amino and carboxy termini oriented intracellularly; B: The
AQP subunits are assembled into a
tetramer with the four sets of B and
E loops constituting four central
water pores.
It was observed that AQP8 can redistribute from the intracellular vesicular compartment to plasma membrane in
hepatocytes stimulated by the cell permeable cAMP analog,
dibutyryl cAMP.15 The cAMP-induced increased levels of
AQP8 on hepatocyte surface are accompanied by an increase in membrane water permeability.15 The microtubule
blocker colchicine specifically inhibits the dibutyryl cAMP
effect on both AQP8 redistribution to cell surface and hepatocyte membrane water permeability,15 providing evidence
for the involvement of microtubules for AQP8 trafficking.
More recent studies in isolated rat hepatocytes show that the
cAMP-mediated hormone glucagon, is also able to induce
the translocation of AQP8 to canalicular membrane.23 Glucagon effect requires activation of the cAMP-dependent protein kinase A and an intact microtubular network. Since the
AQP8 molecules lack consensus protein kinase A phosphorylation sites24 and therefore may not be phosphorilated, the
effect of glucagon is believed not to be directed towards
AQP8 protein itself but rather to microtubule-associated proteins involved in vesicle trafficking. Confocal immunofluorescence microscopy and functional studies in polarized isolated rat hepatocyte couplets14 demonstrated that AQP8 is
specifically targeted to the canalicular plasma membrane domain, which promotes osmotically-driven water secretion.14
Recent detailed immunoelectron microscopy studies
revealed that AQP8 in hepatocytes is also present on
smooth endoplasmic reticulum adjacent to glycogen
granules as well as in some mitochondria.22 These novel
findings suggest the involvement of AQP8 in preserving
cytoplasmic osmolarity during glycogen metabolism and
in mediating mitochondrial volume changes.
In addition to the described short term regulation of hepatocyte AQP8 by vesicle trafficking, the water channel can also
be modulated on the long term basis by modifying its gene
expression. Thus it was recently found that fasting induces a
remarkable down-regulation of hepatic AQP8 mRNA and
protein. Interestingly, the reduction of AQP8 paralleled the
expected depletion of glycogen liver content. The hepatic
AQP8 levels returned to be normal after refeeding.22
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Hepatocyte AQP8 is an N-glycosylated protein of
about 34 kDa. Initial biochemical, immunohistochemical,
and confocal immunofluorescence studies15,16 showed that
AQP8 is largely intracellular in hepatocytes. Immunoelectron microscopy studies localized AQP8 to pericanalicular vesicles as well as to the canalicular plasma membrane.15,21,22
Aquaporin-9 (AQP9)
Hepatocyte AQP9 is an approximately 32 kDa protein.
Immunohistochemical, confocal immunofluorescence as
Annals of Hepatology 3(4) 2004: 130-136 MG
132
sustraídode-m.e.d.i.g.r.a.p.h.i.c
well
as immunoelectron microscopy studies localized
AQP9
on hepatocytes surface, specifically on the sinusoicihpargidemedodabor
dal plasma membrane domain, with no significant intracellular expression.14,19 AQP9 is an aquaglyceroporin, i.e.,
a water channel membrane protein also permeable to certain small uncharged solutes such as urea and glycerol.
This AQP is thought to allow the rapid cellular uptake or
exit of metabolites with minimal osmotic perturbation.
Hepatocyte AQP9 expression was found to be dependent
on the nutritional status.25,26 Thus, AQP9 was observed to
be markedly induced in fasted rats; a state in which glycerol is actively taken up by liver for gluconeogenesis. No
changes in liver AQP9 levels were observed in rats fed
ketogenic or high-protein diets. Hepatocyte AQP9 expression is also affected by the circulating insulin levels.25,26 Hence, liver AQP9 levels were found to be elevated in diabetic rats and decreased after administration of
insulin. The presence of a negative insulin response element in the AQP9 promoter gene25 suggests an insulinmediated suppression on AQP9 transcription. Interestingly, hepatic expression of AQP9 seems to be gender-dependent. Male rats have higher levels of AQP9 protein
and mRNA than female rats.20
Aquaporin-0 (AQP0)
Similar to AQP8, this AQP is mainly localized intracellularly in hepatocytes, although its precise subcellular
localization has not been determined yet. AQP0 seems
not to be regulated by vesicle trafficking,14 based on its
lack of responsiveness to dibutyryl cAMP. The physiological role of AQP0 in hepatocytes remains to be elucidated.
Water transport by hepatocytes: Canalicular bile
formation
Canalicular bile formation is an osmotic secretory process. The biliary excretion of bile salts, via the bile salt
transporter Bsep, glutathione, via the organic anion transporter Mrp2, and HCO3-, via the Cl -/HCO3- exchanger
AE2 are thought to be the major osmotic driving forces
for canalicular bile flow.1 Conceptually, the generation of
bile flow is ultimately dependent on the molecular and
functional expression of these transporters in the canalicular plasma membrane domain as well as on the canalicular membrane water permeability determined by AQPs.
This is assuming that water flows across hepatocyte epithelial barrier by a predominantly transcellular route, with
minimal paracellular contribution. In fact, existing evidence suggests that the transcellular pathway accounts for
most of the water entering the bile canaliculus. Our recent
experimental observations provide additional support for
this view. Studies in polarized hepatocyte couplets
showed that AQP blockers totally prevented osmoticallydriven water transport into bile canaliculus.14 Moreover,
odarobale
FDP
theoretical calculations based on the:rop
osmotic
basolateral
and canalicular membrane water permeability values,
VC
ed AS, cidemihparG
support a predominant
transcellular
route for water movement during choleresis.27 Thus, the transcellular pathway
arapentering the
is thought to account for most of the water
bile canaliculus.
acidémoiB in
arutaretiL
:cihpargideM
As mentioned
the previous
section, AQPs are
sustraídode-m.e.d.i.g.r.a.p.h.i.c
present
in hepatocytes at both apical and basolateral plasma membrane domains as well as in intracellular vesicle
compartments. Two of these AQPs can account for the
water permeability of both hepatocyte plasma membrane
domains, AQP8 modulating mainly the canalicular transport of water, and AQP9 facilitating its basolateral movement. Glucagon (via cAMP) regulates the amount of
canalicular AQP8.
Canalicular bile flow is known to be modulated by
glucagon.28,29 Although the actual osmotic gradients involved in glucagon-induced choleresis are unknown,
these transient gradients are most likely created by the facilitated transport of HCO3- via the canalicular AE2.28,29
There is evidence to suggest that glucagon (via cAMP) is
able to stimulate the microtubule-dependent vesicle trafficking of AE2 to hepatocyte plasma membrane.29,30 This
mechanism, together with direct activation of the exchanger, could account for the bicarbonate-rich choleresis induced by glucagon. Whether AQP8 is packaged
along with AE2 in the same population of vesicles, as has
been observed for AQP1 in cholangiocytes,31 or they are
in separate vesicles that share a common microtubule-dependent, cAMP-related mechanism, needs to be determined. Moreover, as we observed that AQP8 is targeted
to the canalicular plasma membrane domain,14,23 we believe that AQP8 can facilitate the osmotically driven water transport during glucagon-stimulated hepatocyte bile
formation. Thus, our findings in isolated cells may be relevant to the in vivo situation.
Direct assessment of osmotic water permeability in
isolated canalicular and basolateral plasma membrane
vesicles by stopped-flow spectrophotometry,27 indicates
the presence of both lipid and AQP-mediated pathways
for basolateral and canalicular water movement across
hepatocyte plasma membrane barrier. The canalicular
membrane domain has lower water permeability and becomes more permeable to water when hepatocytes are exposed to cAMP-mediated choleretic agonists.
In light of these new observations, it seems plausible
that water can move across hepatocyte membranes by
both lipid- and AQP-mediated pathways. They also suggest that the canalicular plasma membrane domain is rate
limiting for transcellular water transport in hepatocytes,
and that under cAMP-mediated stimulation, this domain
becomes highly permeable to water by insertion of AQP8
water channels facilitating transcellular osmotic water
transport. Thus, hepatocytes are able to modulate their
canalicular membrane water permeability, providing a
molecular mechanism for the efficient coupling of osmot-
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RA Marinelli et al. Liver aquaporins
ically active solutes and water transport during canalicular bile formation. In figure 2, we show a proposed model
for the mechanisms of water transport in hepatocytes.
133
Aquaporin-1 (AQP1)
Rat cholangiocytes express the water channel AQP1,
which is present as a non-glycosylated protein of 28
kDa.32,33 Subcellular fractionation as well as light and immunoelectron microscopy analysis showed that AQP1 is
present mainly in the cholangiocyte apical plasma membrane domain and in an intracellular vesicular pool.31,33,34
Studies in highly purified freshly isolated cholangiocytes
as well as in intact rats, indicate that secretin induces the
Cholangiocyte aquaporins
It has been recently shown that rat cholangiocytes express message and protein for AQP132,33 and AQP4.34 AQP8
was found to be expressed in cholangiocytes at message level,35 but its protein expression needs to be confirmed.
Sinusoid
Glucagon
AQP9
cAMP
PKA
Exocytosis
AQP8
ST
Microtubules
Figure 2. Proposed mechanisms of water transport in hepatocytes. On the left, it is illustrated a hepatocyte in the basal (unstimulated) state. AQP9 is
expressed at the basolateral plasma membrane, while AQP8 is expressed at the canalicular membrane as well as in intracellular vesicles. The canalicular membrane domain has lower water permeability than the basolateral, i.e. it is rate limiting for transcellular water transport. Water secretion by
hepatocyte into the canaliculus occurs mainly transcellularly via lipid and AQP-mediated pathways. On the right, it is illustrated a hormone-stimulated hepatocyte. After binding to its receptor, glucagon, via cAMP, activates protein kinase A which in turn induces the microtubule-dependent canalicular targeting of AQP8-containing vesicles. Canalicular membranes become highly permeable to water by insertion of AQP8. The activation of
preexisting or newly inserted solute transporters (ST), such as the Cl-/HCO3- exchanger AE2, provides the osmotic driving forces for transcellular
water transport into bile canaliculus.
Plasma
Cholangiocyte
Ductal
bile
AE2
Cl
CFTR
AQP4
CFTR
-
AQP1
HCO3
AQP1
Microtubules
Cholangiocyte
-
Cl
-
Water
cAMP
Secretin
Exocytosis
AE2
AQP1
Plasma
AQP1
Water
edigraphic.com
AQP4
Figure 3. Proposed mechanisms of water transport by cholangiocytes. On the left, it is illustrated a cholangiocyte in the basal (unstimulated) state.
AQP4 and AQP1, which are expressed at the basolateral and apical plasma membrane domain respectively, facilitate transcellular AQP-mediated
water transport in duct epithelia. In unstimulated cholangiocytes, AQP1 is mainly located in intracellular vesicles. In a particular population of vesicles, AQP1 colocalizes with CFTR Cl- channels and the Cl-/HCO3- exchanger AE2. On the right, it is illustrated a secretin-stimulated cholangiocyte.
Secretin, via cAMP, triggers the microtubule-dependent apical targeting and exocytic insertion of CFTR, AE2, and AQP1. The efflux of Cl- via CFTR
provides the luminal substrate to drive the extrusion of HCO3- into the lumen via AE2. The presence of HCO-3 and Cl- molecules in the ductal lumen
creates the necessary osmotic force for the movement of water via AQPs into the biliary lumen.
Annals of Hepatology 3(4) 2004: 130-136 MG
134
microtubule-dependent redistribution of AQP1 from intracellular vesicles to the apical plasma membrane domain with a concomitant increase in cholangiocyte membrane water permeability.33,34
AQP1 is expressed in erythrocytes and in fluid transporting epithelia throughout the body at sites of constitutive (not regulated) water transport.4 Liver appears to be
an exception in that AQP1 is present in cholangiocytes in
a pool of AQP1 carrying-vesicles able to traffic to the
plasma membrane in a regulated fashion. Thus, it may be
that a protein that is constitutively expressed in one cell
type is regulated in another. Proteins are under the control
of mechanisms that include interactions between signals
within the protein itself and the cellular sorting machinery. These signals can be differentially interpreted by various cell types.
It was recently found in cholangiocytes a specialized
population of intracellular vesicles containing AQP1 as
well as CFTR Cl- channels and the Cl-/HCO3- exchanger
AE2. After exposure of cholangiocytes to dibutyryl AMP
in vitro or after secretin infusion in vivo, these three proteins co-redistribute to the apical cholangiocyte plasma
membrane.31 It is expected that this process plays a key
role in hormone-induced ductal bile secretion.
Aquaporin-4 (AQP4)
Cholangiocyte AQP4 is a protein of approx. 31 kDa.34
AQP4 is not responsive to secretin and it seems to be constitutively localized to the cholangiocyte basolateral
membrane, mediating water transport across that plasma
membrane domain.
Water transport by cholangiocytes: Ductal bile
formation
Cholangiocytes account for only 3% to 5% of the liver
cell population; nevertheless, they provide a large surface
area for transport between blood to bile and play a significant role in bile secretion producing as much as 40% of
total bile volume in some species.37 These observations
suggest that the amount of transcellular water movement
across an individual cholangiocyte is potentially up to 10
times greater than across an individual hepatocyte.
AQPs are present in cholangiocytes in both apical and
basolateral plasma membrane domains as well as in intracellular vesicle compartments. Thus, AQPs can account
for the water permeability of both cholangiocyte membrane domains, AQP1 modulating mainly the apical
transport of water, and AQP4 facilitating its basolateral
movement. Such basolateral transport of water would allow the relative isosmolar status of the cell to be maintained during ductal bile formation and is consistent with
the intimate physical association between the basolateral
domain of cholangiocytes and the peribiliary vascular
plexus, a mesh-like arrangement of blood vessels that sur-
round the bile duct and from which the fluid component
of bile originates. High expression of AQP1 in peribiliary
vascular endothelia34 may reflect an important functional
role of this AQP in facilitating water transport from plasma to bile across cholangiocytes.
It is well known that ductal bile secretion is regulated
by secretin. We have recently demonstrated that secretin
stimulated ductal bile secretion is associated with the exocytic insertion of AQP1, normally sequestered in cytoplasmic vesicles, into the cholangiocyte apical plasma
membrane. Thus, inhibitory studies in isolated rat and
mouse bile duct segments suggest that AQP1 plays a key
role in osmotically-driven apical water secretion during
hormone-regulated ductal bile formation. Indeed, studies
in perfused rat bile duct segments in which AQP1 was silenced in vitro using small interfering RNA technology
provided additional evidence for a key role for AQP1 in
ductal bile formation.38
Although secretin is well known to stimulate ductal bile
secretion by interacting with specific cAMP-coupled receptors on the basolateral domain of cholangiocytes,2 little
information is available on the stimulus-secretion coupling
mechanisms. Based on current knowledge, we propose a
model for coupling solute and water secretion in cholangiocytes. In this updated model, secretin is proposed to stimulate the exocytic insertion of vesicles containing AQP1,
CFTR Cl- channels, and AE2 (i.e., the Cl-/HCO-3 exchanger) into the apical cholangiocyte membrane. The efflux of
Cl- via CFTR provides the luminal substrate to drive the
extrusion of HCO3- into the lumen via AE2. The presence
of HCO-3 and Cl- molecules in the ductal lumen creates the
necessary osmotic force for the movement of water via
AQPs into the biliary lumen. The efflux of Cl- would also
generate a negative intraluminal potential leading to Na+
and K+ movement through a paracellular pathway; however, the degree to which water follows the efflux of these
two ions via a paracellular route is unclear.
It is not known whether a substantial fraction of transepithelial water flow across the tight junctions between cholangiocytes (i.e., the paracellular route) contributes to ductal bile formation. Using established in vitro physiologic
models suitable for addressing this question (i.e., enclosed
polarized and microperfused bile ducts isolated from normal rats and mice), we observed that osmotically induced
transepithelial water movement into the lumen of bile duct
units was inhibited by DMSO or HgCl2 (agents that inhibit
AQP-mediated water transport) but not by protamine (an
agent that alters paracellular water transport).39,40 These
data suggest that transepithelial water transport across biliary epithelia occurs principally via water channels and not
via a paracellular pathway. Calculated Pf values demonstrating rapid water movement across the biliary epithelia
in rat microperfused intrahepatic bile ducts also support
this concept.41 Taken together, these data suggest that water transport by intrahepatic bile ducts is predominantly
transcellular and AQP-mediated.
edigraphic.com
RA Marinelli et al. Liver aquaporins
Intrahepatic bile ducts not only secrete but also absorb
water.2,3 It was found that enclosed polarized intrahepatic
bile duct units exposed to hypertonic buffers rapidly decreased their luminal area, reflecting net water absorption. Substantial water movement from lumen to bath was
observed if a net outward osmotic gradient was established in microperfused rat and mouse bile duct units.39-41
Addition of glucose to the perfusate of isolated microperfused rat bile duct units was also associated with water
absorption (42). In these models, water absorption by biliary epithelia was sensitive to AQP inhibitors, suggesting
the involvement of water channels.39,40 AQPs 1 and 4 are
the most likely candidates to provide channel-mediated
water absorption by biliary epithelia.
Implications of AQPs in liver disease
Bile secretion is compromised in several illnesses, and
the pathogenesis of bile secretory failure cannot be understood without a thorough knowledge of how bile is generated. It is known that bile secretion by hepatocytes and
cholangiocytes results from the coordinated interactions
of several solute membrane-transport systems. As detailed above, recent cumulative evidence indicates that
AQP water channels play a key physiological role in
canalicular and ductal bile formation. Hence, it is conceivable that defective AQP membrane expression may
lead to alterations of normal bile physiology. Currently,
there is no conclusive evidence indicating that derangements of normal AQP function are causative of bile secretory dysfunction. Nevertheless, abnormal expression and
trafficking of AQPs were found to be present in some
pathological conditions in which altered bile secretion occurs. We have recently demonstrated,21 by using immunoblotting, immunohistochemical and immunoelectron microscopy studies, a defective expression of hepatocyte
AQP8 in rats with biliary obstruction (i.e., extrahepatic
cholestasis). Northern blotting experiments indicated that
the levels of AQP8 mRNA were not decreased, suggesting a normal message transcription.21 Thus, although the
precise mechanism for AQP8 protein reduction has not
been elucidated yet, it seems to be posttranscriptional. It
may involve a defective synthesis and/or degradation of
AQP8 protein.
We also found that the vesicular translocation of
AQP8 to the hepatocyte plasma membrane was compromised in extrahepatic cholestasis.21 As mentioned in previous sections, the microtubules seem to be central for the
hormone-regulated vesicle trafficking of liver AQPs. It is
known that hepatocyte microtubules undergo significant
changes after bile duct ligation,43 which is thought to disturb vectorial vesicular transport of some canalicularly
targeted transporters.43 Thus, defective translocation of
AQP8 in extrahepatic cholestasis may be due, at least in
part, by alterations in the microtubular network. An alteration of the hepatic cAMP level does not seem to be in-
135
volved in the defective AQP8 translocation, since cAMP
was found not to be altered by bile duct ligation.44
The finding that AQP8 expression and trafficking are
defective in extrahepatic cholestasis suggests, for the first
time, that AQP water channels are involved in the development of bile secretory dysfunction.
Alterations in the functional expression of renal AQPs
have also been involved in liver pathology. For example,
the levels of AQP2, the vasopressin-sensitive AQP of kidney collecting tubular cells,45 were found to be altered in
experimental models of hepatic cirrhosis.45 AQP2 has
been observed upregulated in decompensated states of
liver cirrhosis which are characterized by sodium retention, edema, and ascites.46,47 It is though that the increased
levels of AQP2 would in turn participate in the development of water retention. Interestingly, changes in expression of AQP2 protein levels vary considerably between
fundamentally different experimental models of cirrhosis.
Thus, AQP2 is downregulated in compensated cirrhosis,
characterized by peripheral vasodilatation and increased
cardiac output but not water retention.48,49 It has been suggested that the decreased of AQP2 may represent a compensatory mechanism to prevent development of water
retention.
Conclusion
The understanding of the physiological mechanisms of
bile formation by hepatocytes and cholangiocytes is rapidly evolving because of the recent discovery of the AQP
water channels. This knowledge is expected to shed new
light on the molecular pathogenesis of certain liver diseases with compromised bile secretion.
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